Pulse photodynamic treatment of photodamaged skin

ABSTRACT

The present invention is related to a pulse photodynamic therapy (or pulse PDT) treatment of photodamaged skin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 16/896,097, filed Jun. 8, 2020, which is a Continuation Applicationof U.S. application Ser. No. 15/103,603, filed Jun. 10, 2016, issued asU.S. Pat. No. 10,716,855, which is a National Stage Application ofPCT/EP2014/076796, filed Dec. 5, 2014, and designating the United States(published on Jun. 25, 2015, as WO 2015/091037 A1), which claimspriority under 35 U.S.C. § 119 to European Patent Application No.14162516.0, filed Mar. 28, 2014, and European Patent Application No.13306826.2, filed Dec. 20, 2013, each hereby expressly incorporated byreference in its entirety and each assigned to the assignee hereof.

FIELD OF THE INVENTION

The present invention is related to a pulse photodynamic therapy (orpulse PDT) treatment of photodamaged skin.

BACKGROUND OF THE INVENTION

Photodamaged or sun damaged skin occurs with exposure to sunlight (UVAor UVB). Twenty five percent of our lifetime exposure occurs before theend of our teenage years. Photodamage is considered to be the structuraland functional deterioration of chronically sun-exposed skin.Keratinocytes, melanocytes, fibroblasts and endothelial cells arealtered by UV radiation resulting in changes in skin texture, alteredskin tightness and thickness, sallowness, dyschromia, wrinkles,telangiectasias, erythema, sebaceous gland hypertrophy and epithelialatypia or dysplasia (Baumann L. J Pathol. 2007 January; 211(2):241-51.Skin ageing and its treatment; Han A. Photoaging. Dermatol Clin. 2014:291-9).

For the most part these changes occur most frequently on areas ofchronic exposure including the face, ears, neck, backs of the hands,chest, arms and legs. Buttocks or upper inner arms are often preservedand pristine emphasizing the difference between chronological aging andphotoaging.

As the manifestations of photodamage are numerous many options oftreatment are proposed, such as: chemical peels, dermabrasion,injectable fillers, botulinum toxin and surgery, as well as topicaltreatments such as the use of retinoids and lately ablative andnon-ablative resurfacing lasers carbon dioxide laser, ND-Yag,Q-switched, KTP and pulsed-dye lasers and intensed pulsed light (IPL)),radiofrequency and photodynamic therapy (PDT) as alternatives. (ShambanA T. Current and new treatments of photodamaged skin. Facial Plast Surg.2009 December; 25(5):337-46).

Nowadays photodynamic therapy (PDT), a recognized and approved treatmentfor nonmelanoma skin cancers and their precursors such as actinickeratosis, as well as for other organ and mucosal epithelial conditions,sees its indications enlarging to inflammatory or infectious conditions(e.g. psoriasis, acne, leishmaniosis). PDT involves the application of aphotosensitizing (photochemotherapeutic) agent to the affected area ofthe body, followed after an incubation period to the exposure to aphotoactivating light that will convert the photosensitizing agent intoa cytotoxic form, followed by necrosis and apoptosis of the targettissue

A range of photosensitizing agents is known, including the psoralens,the porphyrins (e.g. Photofrin (Registered trademark)), the chlorins andthe phthalocyanins. Amongst the most clinically useful photosensitizingagents known in the art, however, are 5-aminolevulinic acid and itsderivatives, for example esters such as 5-ALA esters. These, through anintra cellular metabolism will be converted by the haem biosyntheticpathway predominantly to protoporhyrin IX (PpIX).

The mechanism of action of PDT relies on intracellular porphyrins(including PpIX) that are photoactive, fluorescing compounds and, uponlight activation in the presence of oxygen, singlet oxygen is formedwhich causes damage to cellular compartments, in particular themitochondria. Light activation of accumulated porphyrins leads to aphotochemical reaction and thereby phototoxicity to the light-exposedtarget cells.

Although PDT is clinically useful in the treatment of a wide range ofdiseases, a major drawback of such treatment is the concomitantside-effects, particularly at the treatment site. These often includeinflammation such as erythema, swelling, edema, burning, itching,exfoliation, hyperpigmentation and prolonged irritation andhypersensitivity after treatment. Such side-effects are particularlyundesirable when the treatment site is the face, scalp or neck.

A need still therefore exists for alternative PDT methods especiallyusing natural daylight with reduced or no undesirable side effects (e.g.inflammation, pain, etc.) as well as lower downtime but which have hightherapeutic efficacy.

SUMMARY OF THE INVENTION

The present invention relates to a PDT treatment of photodamaged skin,comprising subjecting the skin of a subject in need thereof to apre-treatment such as mechanical ones (like dermoabrasion (e.g. withsand paper) or microneedling (e.g. with a dermaroller)) or chemical oneslike peeling. The inventors have surprisingly found that such mechanicalpre-treatment is as efficient in cosmetically or therapeuticallytreating photodamaged skin without, or with reduced, adverse effectsobserved with a pretreatment implemented with ablative fractional laser.The PDT then comprises applying onto said skin a photosensitizer, inparticular 5-MAL. In a representative embodiment, the photosensitizer isapplied for a duration comprised between 4 minutes to 4 hours, inparticular between 15 minutes and 3 hours.

The present inventors have also surprisingly found that application of aphotosensitizer for a shorter time period that is classicallyimplemented in a PDT, allows the implementation of a PDT as efficient asin the case where the photosensitizer is used for a longer period oftime, with greatly reduced side effects usually observed in the priorart PDT protocols.

Therefore, the invention also relates to a PDT treatment of photodamagedskin, comprising administering to a subject in need thereof aphotosensitizer, in particular 5-MAL, for a short duration and thenremoving the photosensitizer from the skin surface. This PDT protocol isalternatively designated pulse-PDT herein.

Representative photosensitizers include preferably 5-aminolevulinic acid(5-ALA) and derivatives (e.g. an ester) of 5-ALA, more preferably 5-ALAmethyl ester (or 5-MAL), or a pharmaceutically acceptable salt thereof.In the present uses and methods, photoactivation is achieved by naturalor artificial light. In a particular embodiment, the PDT comprises:

-   -   (a) optionally, preparing the area of skin to be treated with        the appropriate pre-treatment, in particular a mechanical        pretreatment such as a curettage, dermoabrasion or        micro-needling (or micro perforation),    -   (b) administering to said animal a composition comprising said        photosensitizer, in particular for a short duration; and    -   (c) photoactivating said photosensitizer.

In a particular embodiment, the invention implements a pulse-PDTtreatment, comprising administering to a subject in need thereof aphotosensitizer, in particular 5-MAL, for a short duration and thenremoving the photosensitizer from the skin surface. Photoactivation isthen carried out as described throughout the present application. Thepulse-PDT treatment of the invention ensures high intracellular PPIX andlow extracellular PPIX. Excess amounts of PPIX formation during andafter the end of the treatment are thus avoided. In particular, theinventors show that the pulse-PDT treatment of the invention shows lessinflammation with unchanged efficacy.

According to an embodiment, the pulse time during which thephotosensitizer is let on the skin is comprised between 5 and 120minutes. According to a preferred embodiment, the pulse time duringwhich the photosensitizer is let on the skin is comprised between 15 and60 minutes, in particular between 20 and 40 minutes. In a furtherparticular embodiment, the photosensitizer is administered for about 30minutes (e.g. for 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 minutes,more particularly during 30 minutes).

DETAILED DESCRIPTION OF THE INVENTION

By the term “animal” is meant herein any human or non-human being.Preferred animals for treatment in accordance with the invention arehumans.

In a particular embodiment, the subject is a male or female humansubject. In another particular embodiment, the subject is of theFitzpatrick I, II, III, IV, V or VI skin type. In a further embodiment,the subject is of the I, II or III skin type, more particularly of theII or III skin type.

In another embodiment, the subject's skin Dover's global photodamagescore before treatment if of 2, 3 or 4, in particular of 2 or 3.

Photodamage occurs when skin of an organism is exposed to ultraviolet(especially ultraviolet-B, UV-B), leading to skin damage. Exposure to UVradiation will accelerate the accumulation of ROS and free radicals inskin cells, increase oxidative stress to the skin cells and induceexpression of matrix metalloproteinases (MMP), thereby resulting inoxidative photodamage. Molecular changes of photoaging are reflected bythe alteration of keratinocytes, melanocytes fibroblasts and endothelialcells by UV. The morphologic changes are: thickening of epidermis,increased melanocyte density and epidermal melanin, abnormal elastictissue, increased collagen degradation, thickening of the vascularwalls.

All these changes are translated by clinical signs such as dryness,sallowness, fine and severe wrinkling, irregular dark/lightpigmentation, laxity, elastosis (yellow, cobblestoned effect),telegiectasia, sebaceous gland hyperplasis and in advanced cases,premalignant and malignant lesions care occur. Chronic actinic damage offacial skin. Clin Dermatol. 2014 November-December; 32(6):752-62).

Photosensitizers

Use of 5-ALA (5-amino-4-oxo-pentanoic acid, otherwise known as5-aminolevulinic acid) and derivatives of 5-ALA in PDT is well known inthe scientific and patent literature (see, for example, J. C. Kennedy etal., J. Clin. Laser Med. Surg. (1996) 14: 289-304, U.S. Pat. Nos.5,079,262, 5,211,938, 5,234,940, 5,422,093, 6,034,267, WO91/01727,WO96/28412, WO2005/092838 and WO2006/051269). 5-ALA and all suchderivatives of 5-ALA, as well as their pharmaceutically acceptablesalts, are suitable for the uses and methods herein described.

The 5-ALA derivatives useful in accordance with the invention may be anyderivative of 5-ALA capable of forming protoporphyrin IX (PpIX) or anyother photosensitizer (e.g. a PpIX derivative) in vivo. Typically, suchderivatives will be a precursor of PpIX or of a PpIX derivative (e.g. aPpIX ester) and which are therefore capable of inducing an accumulationof PpIX at the site to be treated following administration in vivo.Suitable precursors of PpIX or PpIX derivatives include 5-ALA prodrugswhich might be able to form 5-ALA in vivo as an intermediate in thebiosynthesis of PpIX or which may be converted (e.g. enzymatically) toporphyrins without forming 5-ALA as an intermediate. Esters of5-aminolevulinic acid and N-substituted derivatives thereof arepreferred photosensitizers for use in the invention. Those compounds inwhich the 5-amino group is unsubstituted (i.e. the ALA esters) areparticularly preferred. Such compounds are generally known and describedin the literature (see, for example, WO96/28412, WO02/10120 andWO2005/092838 to PhotoCure ASA). Esters of 5-aminolevulinic acid withsubstituted or unsubstituted alkanols, i.e. alkyl esters are especiallypreferred photosensitizers for use in the invention. In particular,5-MAL and 5-MAL derivatives are particularly preferred. Examples ofuseful derivatives include those of general formula I:R² ₂N—CH₂COCH₂—CH₂CO—OR¹  (I)

Wherein:

R¹ represents a substituted or unsubstituted straight, branched orcyclic alkyl group (e.g. a substituted or unsubstituted straight-chainedalkyl group); and each R² independently represents a hydrogen atom or anoptionally substituted alkyl group, e.g. a group R¹; andpharmaceutically acceptable salts thereof.

As used herein, the term “alkyl”, unless stated otherwise, includes anylong or short chain, cyclic, straight-chained or branched aliphaticsaturated or unsaturated hydrocarbon group. The unsaturated alkyl groupsmay be mono- or polyunsaturated and include both alkenyl and alkynylgroups. Unless stated otherwise, such groups may contain up to 40 atoms.However, alkyl groups containing up to 30, preferably up to 10,particularly preferably up to 8, especially preferably up to 6, e.g. upto 4 carbon atoms, for example 1, 2, 3 or 4 carbon atoms, are preferred.

The substituted alkyl R¹ and R² groups may be mono or poly-substituted.

Suitable substituents may be selected from hydroxy, alkoxy, acyloxy,alkoxycarbonyloxy, amino, aryl, nitro, oxo, fluoro, —SR3, —NR³ ₂ and—PR³ ₂ groups, and each alkyl group may be optionally interrupted by oneor more —O—, —NR³—, —S— or —PR³— groups, in which R³ is a hydrogen atomor a C₁₋₆ alkyl group).

Preferred substituted alkyl R¹ groups include those carrying one or moreoxo groups, preferably straight-chained C₄₋₁₂ alkyl (e.g. C₈₋₁₀ alkyl)groups substituted by one, two or three (preferably two or three) oxogroups. Examples of such groups include 3,6-dioxa-1-octyl and3,6,9-trioxa-1-decyl groups.

Particularly preferred for use in the invention are those compounds offormula I in which at least one R² represents a hydrogen atom. Inespecially preferred compounds each R² represents a hydrogen atom.

Compounds of formula I in which R¹ represents an unsubstituted alkylgroup (preferably C₁₋₈ alkyl, e.g. C₁₋₆ alkyl) or an alkyl group (e.g.C₁₋₂ alkyl, especially C₁ alkyl) substituted by a substituent ashereinbefore defined (e.g. by an aryl group such as phenyl or by analkoxy group such as methoxy) are also preferred.

Unsubstituted alkyl groups which may be used in the invention includeboth branched and straight-chained hydrocarbon groups. Compounds offormula I in which R¹ is a C₄₋₈, preferably a C₅₋₈, straight chain alkylgroup which is branched by one or more C₁₋₆ (e.g. C₁₋₂ alkyl) groups arepreferred. Representative examples of suitable unsubstituted branchedalkyl groups include 2-methylpentyl, 4-methyl pentyl, 1-ethylbutyl and3,3-dimethyl-1-butyl. 4-methylpentyl is particularly preferred.

Compounds of formula I in which R¹ is a C₁₋₁₀ straight-chained alkylgroup are also preferred. Representative examples of suitableunsubstituted alkyl groups include methyl, ethyl, propyl, butyl, pentyl,hexyl and octyl (e.g. n-propyl, n-butyl, n-pentyl, n-hexyl and n-octyl).Hexyl, especially n-hexyl, is a particularly preferred group. Methyl isalso particularly preferred.

Also preferred for use in the invention are those compounds of formula Iin which R¹ represents a C₁₋₂ alkyl group (preferably a C₁ alkyl group)optionally substituted by an aryl group.

Still further preferred for use in the invention are those compounds offormula I in which R¹ represents an alkyl group (e.g. C₁₋₂ alkyl,especially C₁ alkyl) substituted by an aryl group (e.g. phenyl).Preferred substituted alkyl R¹ groups which may be present in compoundsof formula I include C₁₋₆ alkyl, preferably C₁₋₄ alkyl, particularlypreferably C₁ or C₂ alkyl (e.g. C₁ alkyl) substituted (preferablyterminally substituted) by an optionally substituted aryl group.

By an “aryl group” is meant a group which is aromatic. Preferred arylgroups comprise up to 20 carbon atoms, more preferably up to 12 carbonatoms, for example, 10 or 6 carbon atoms.

Aryl groups which may be present in the compounds of the invention maybe heteroaromatic (e.g. 5-7 membered heteroaromatics) but are preferablynonheteroaromatic. By “non-heteroaromatic” is meant an aryl group havingan aromatic system comprising electrons originating solely from carbonatoms. Preferred aryl groups include phenyl and napthyl, especiallyphenyl. In preferred compounds for use in the invention one or two arylgroups may be present, preferably one.

Aryl groups which may be present in the compounds of the invention mayoptionally be substituted by one or more (e.g. 1 to 5), more preferablyone or two, groups (e.g. one group). Preferably the aryl group issubstituted at the meta or para position, most preferably the paraposition. Suitable substituent groups may include haloalkyl (e.g.trifluoromethyl), alkoxy (i.e. —OR groups wherein R is preferably a C₁₋₆alkyl group), halo (e.g. iodo, bromo, more especially chloro andfluoro), nitro and C₁₋₆ alkyl (preferably C₁₋₄ alkyl). Preferred C₁₋₆alkyl groups include methyl, isopropyl and t-butyl, particularly methyl.Particularly preferred substituent groups include chloro and nitro.Still more preferably the aryl group is unsubstituted.

In a further preferred aspect the invention provides the use of aphotosensitiser which is a compound of formula I wherein R¹ representsan aryl substituted C₁₋₄ alkyl group (preferably C₁₋₂, e.g. C₁),preferably wherein said aryl group comprises up to 20 carbon atoms (e.g.up to 12 carbon atoms, especially 6 carbon atoms) and is itselfoptionally substituted, and each R² is as hereinbefore described.

Preferred compounds for use in the invention include methyl ALA ester,ethyl ALA ester, propyl ALA ester, butyl ALA ester, pentyl ALA ester,hexyl ALA ester, octyl ALA ester, 2-methoxyethyl ALA ester,2-methylpentyl ALA ester, 4-methylpentyl ALA ester, 1-ethylbutyl ALAester, 3,3-dimethyl-1-butyl ALA ester, benzyl ALA ester,4-isopropylbenzyl ALA ester, 4-methylbenzyl ALA ester, 2-methylbenzylALA ester, 3-methylbenzyl ALA ester, 4-[t-butyl]benzyl ALA ester,4-[trifluoromethyl]benzyl ALA ester, 4-methoxybenzyl ALA ester,3,4-[dichloro]benzyl ALA ester, 4-chlorobenzyl ALA ester, 4-fluorobenzylALA ester, 2-fluorobenzyl ALA ester, 3-fluorobenzyl ALA ester,2,3,4,5,6-pentafluorobenzyl ALA ester, 3-nitrobenzyl ALA ester,4-nitrobenzyl ALA ester, 2-phenylethyl ALA ester, 4-phenylbutyl ALAester, 3-pyridinyl-methyl ALA ester, 4-diphenyl-methyl ALA ester andbenzyl-5-[(1-acetyloxyethoxy)-carbonyl]amino levulinate.

Still further preferred compounds for use in the invention includemethyl ALA ester, ethyl ALA ester, 2-methoxyethyl ALA ester, benzyl ALAester, 4-isopropylbenzyl ALA ester, 4-methylbenzyl ALA ester,2-methylbenzyl ALA ester, 3-methylbenzyl ALA ester, 4[t-butyl]benzyl ALAester, 4-[trifluoromethyl]benzyl ALA ester, 4-methoxybenzyl ALA ester,3,4[di-chloro]benzyl ALA ester, 4-chlorobenzyl ALA ester, 4-fluorobenzylALA ester, 2-fluorobenzyl ALA ester, 3-fluorobenzyl ALA ester,4-nitrobenzyl ALA ester, 2-phenylethyl ALA ester, 4-phenylbutyl ALAester, 3-pyridinyl-methyl ALA ester, 4-diphenyl-methyl ALA ester andbenzyl-5-[(1-acetyloxyethoxy)-carbonyl]amino levulinate.

Particularly preferred compounds for use in the invention include methylALA ester, hexyl ALA ester and benzyl ALA ester, especially methyl ALAester.

The compounds for use in the invention may be prepared by anyconventional procedure available in the art (e.g. as described inWO02/10120 to PhotoCure ASA). For example, esters of 5-ALA may beprepared by reaction of 5-ALA with the appropriate alcohol in thepresence of acid. Alternatively compounds for use in the invention maybe available commercially (e.g. from Photocure ASA, Norway).

Other photosensitizers that can be used in the field of the presentinvention can be 1, 1′ bis (2 ethyl 1, 3 dioxolan 2 yl) cryptocyanine, 3carbethoxypsoralen, 4, 4′, 6 trimethylangelicin, 4′ aminomethyl 4, 5′, 8trimethylpsoralen, 4′ hydroxymethyl 4, 5′, 8 trimethylpsoralen, 5methylangelicin, aminolevulinic acid hexyl ester, aminolevulinic acidmethyl ester, amotosalen, angelicin, bacteriochlorin, benzoporphyrinderivative, bergapten, chloroaluminum phthalocyanine, etiopurpurin,fimaporfin, gadolinium texaphyrin, hematoporphyrin derivative,hypocrellin A, hypocrellin B, lemuteporfin, lutetium texaphyrin,merocyanine, methoxsalen, motexafin, musk ambrette, padeliporfin,padoporfin, photofrin, photofrin I, photofrin II, phthalocyanine,phthalocyanine aluminum, phthalocyanine derivative, phthalocyanine zinc,psoralen, psoralen derivative, rostaporfin, talaporfin, temoporfin,tetrakis (3 hydroxyphenyl) chlorin, tetrakis (4 sulfophenyl) porphine,tetraphenylporphyrin, tetraphenylporphyrin derivative,tetrasulfophthalocyanine, tetrasulfophthalocyanine aluminum,tetrasulfophthalocyanine chloroaluminum, trimethylpsoralen, trioxysalen,verdin derivative, verteporfin.

Photoactivation

According to the present invention, photoactivation is achieved byeither an artificial or natural light source. In a preferred embodiment,photoactivation of the photosensitizer is achieve by LED or sunlight.

Penetrations Enhancers or Pre-Treatment

Skin penetration enhancers as well as skin pre-treatment for enhancingpenetration of drugs and chemicals have been developed to improvebioavailability. One action among others of these enhancers orpre-treatment procedures is to decrease the skin barrier resistance.These enhancing penetration procedures can be classified as mechanical,physical and chemical pre-treatments.

The table below is a non-limitative list of suchpre-treatment/penetration enhancers that may be used according to theinvention.

Mechanical Physical Chemical Skin preparation pad Ablative (carbondioxide ) and Superficial peelings (alfa- (sandpaper) non-ablativelasers (fractional hydroxy acids, trichloracetic Microdermabration nonablative carbon dioxide, acid, Jessner solution) microneedlingerbiumdoped yttrium aluminium garnet (Er:YAG), IPL) Curettage Retinoids(tretinoin, tape- stripping adapalene, tazarotene) pan- scrubber Acidazelaic exfoliating scrub Vitamin D3 derivates compress rubbingLight Sources—Artificial

Electroluminescence (EL) is an optical and electrical phenomenon inwhich a material emits light in response to the passage of an electriccurrent or to a strong electric field. This is distinct from black bodylight emission resulting from heat (incandescence), from a chemicalreaction (chemiluminescence), sound (sonoluminescence), or othermechanical action (mechanoluminescence).

Among the electroluminescence sources, LED (Light emitting diodes) lampsare well known and preferred as artificial light source in the presentinvention. A LED lamp (LED light bulb) is a solid-state lamp that useslight-emitting diodes (LEDs) as the source of light. The LEDs involvedmay be conventional semiconductor light-emitting diodes, organic LEDs(OLED), or polymer light-emitting diodes (PLED) devices.

The LED lamps used in the examples hereafter are defined by somecharacteristics like wavelength (in nm), power of the LED (irradiance inmW/cm²) energy of the LED (in J/cm²). Such particular features areprovided below.

Light Sources—Natural

This aspect of the invention includes photoactivation with eithernatural sunlight or any light source which provides artificial sunlight(i.e. the entire range from UV to IR). Use of natural sunlight as thelight source has the advantage that the animal being treated is free toleave the clinical environment where treatment is normally conducted.

Light Sources—Intensity

In the uses and methods of the invention, photoactivation may beachieved using light sources known in the art. Methods for theirradiation of different areas of the body, e.g. by lamps or lasers arewell known in the art (see for example Van den Bergh, Chemistry inBritain, May 1986 p. 430-439). The wavelength of light used forirradiation may be selected to achieve a more efficaciousphotosensitizing effect. The most effective light is light in thewavelength range 300-800 nm, typically within the 400-700 nm range.

Irradiation with an artificial light is preferably performed for 1 to 30minutes, preferably for 1 to 15 minutes, more preferably from 5 to 10minutes, preferably for 5 minutes, depending on the light dose andfluence rate. A single irradiation may be used or alternatively a lightsplit dose in which the light dose is delivered in a number offractions, e.g. a 1 to 10 minutes between irradiations, may be used.

Photoactivation with natural light is preferably done for a durationbetween 5 minutes and 4 hours, in particular for a duration of 2 hours.In a particular embodiment, a sunscreen is applied to the sun-exposedarea including the treatment area in both groups during naturaldaylight-PDT, to avoid sunburn.

Treatment of the Skin According to the Invention

The methods and uses of the invention may involve pretreatment of theskin. As the stratum corneum acts as a barrier that limits thepenetration of substances through the skin, the purpose of apretreatment (enhancer) is to favor the absorption of thephotosensitizer to the target tissue and thus a higher efficacy.Enhancers may comprise mechanical, physical or chemical preparation ofthe skin e.g. microdermabrasion (particularly with an adapted skinpreparation pad, sandpaper), microneedling, tape-stripping,pan-scrubber, exfoliating scrub, compress rubbing, non ablative lasersat a low-energy delivery and chemical procedures such as superficialpeelings (Retinoids (tretinoin, adapalene, tazarotene), Acid azelaic,Vitamin D3 derivates). For example, the pretreatment may comprise amechanical pretreatment of the skin. Representative mechanicaltreatments include curettage, dermoabrasion (in particular with anadapted sandpaper or micro-needling (or micro-perforation) beforeapplication of the photosensitizer. In a particular embodiment, thepretreatment includes perforation of the skin using an adapted devicesuch as a micro-needle device, for example a dermaroller.

The skin rejuvenating effects of PDT using an artificial light sourcefor photodamaged skin have been documented in several clinical trials.(Jang JID 2013, Szeimies BJD 2012, Sakamoto BJD 2011, Choi J Y. JDermatol Science. 2010, Orringer Arch Dermatol 2008, Issa Dermatol Surg2010, Zane. Laser Surg Med 2007, Bagazgoita BJD 2011). It was raised theissue that PDT with artificial light sources for photorejuvenation canbe a painful procedure and therefore often requires specific painmanagement.

Daylight mediated PDT appears as the ideal procedure addressing to allsigns of epidermal and dermal actinic damage with lack of discomfort,lack of pain during therapy, possibility to treat large areas (asdemonstrated by several trials showing the same efficacy level of bothprocedures, using daylight or an artificial source for clearing actinickeratosis with significantly lower pain score, leading to fewer relatedadverse (Wiegell JEADV 2011).

The use of an enhancer, such as a skin pretreatment as described above,in particular a mechanical pretreatment, more particularly the use ofdermabrasion or microneedling, and more particularly using a sandpaper,associating natural light could provide better effects in photodamagedskin with lower side effects.

The methods and uses of the invention may also be carried out with orwithout occlusion, more preferably with occlusion.

The photosensitizer may be applied for a duration of between 5 minutesto 4 hours, in particular between 15 minutes to 3 hours, in particularbetween 30 minutes and 2 hours. In a particular embodiment, thephotosensitizer may be applied as a pulse therapy for the time periodsprovided above, for example for a duration of about 30 minutes. Theinventors herein show that such a pulse therapy has the advantage ofbeing as efficient as therapy with longer exposures, but with less PPIXproduced, thereby preventing side effects associated with PPIX.

In a particular embodiment, the treatment comprises:

-   -   (a) optionally, preparing the area of skin to be treated with        the appropriate pre-treatment, for example a curettage, a        dermoabration or microneedling (micro perforation), in        particular a perforation with an adapted micro-needling device        such as a dermaroller;    -   (b) administering to said animal a composition comprising said        photosensitizer, in particular for a short period of time;    -   (c) photoactivating said photosensitizer; and    -   (d) optionally, removing the photosensitizer.

In an embodiment of the invention, the natural daylight photodynamictherapy (PDT) on an animal comprises:

-   -   a) optionally, preparing the area of skin to be treated with the        appropriate pre-treatment, for example a curettage, a        dermoabration or microneedling (micro perforation), in        particular a perforation with an adapted micro-needling device        such as a dermaroller;    -   b) administering to said animal a composition comprising said        photosensitizer for a duration between 5 min to 240 minutes;    -   c) photoactivating said photosensitizer for a duration between 1        to 15 minutes with artificial light or 0.5 hour to 3 hours with        natural light; and    -   d) Optionally, removing the photosensitizer.

In a more preferred embodiment of the invention the use of aphotosensitizer in natural daylight photodynamic therapy (PDT) on ananimal comprises:

-   -   a) optionally, preparing the area of skin to be treated with the        appropriate pre-treatment, for example a curettage, a        dermoabration or microneedling (micro perforation), in        particular a perforation with an adapted micro-needling device        such as a dermaroller;    -   b) administering to said animal a composition comprising said        photosensitizer for a duration between 15 min to 180 minutes;    -   c) photoactivating said photosensitizer for a duration between        0.5 hour to 2 hours with natural light; and    -   d) optionally, removing the photosensitizer.

In a more preferred embodiment of the invention the photosensitizer foruse in photodynamic therapy (PDT) on an animal comprises

-   -   a) optionally, preparing the area of skin to be treated with the        appropriate pre-treatment, for example a dermoabration        (sand-paper) or microneedling (micro perforation), in particular        a dermoabration with an adapted sand-paper device such as        silicone carbide sand paper;    -   b) administering to said animal a composition comprising said        photosensitizer for a duration of 30 minutes; and    -   c) photoactivating said for a duration of at least 2 hours with        natural light; and    -   d) optionally, removing the photosensitizer.

Any of the above particular or preferred embodiments may comprise a stepof mechanically pretreating the skin as described above, before the stepof applying the photosensitizer on the skin.

In a particular embodiment of the invention, PDT of photodamaged skin isapplied to treat any one or all of the manifestation of photodamage ofthe skin: texture (tactile roughness), sallowness (pale skin), wrinkles(fine lines), mottled pigmentation, dyspigmentation(hypo-/hyperpigmentation including solar lentigines), facial erythemaand elastosis, with a special interest in fine wrinkles and pigmentarytroubles. (including advanced aging or wrinkling, uneven or “pebbly”skin, irregular pigmentation, small blood vessels or red markings(telangiectasias), freckles (ephilides), “liver spots” and “age spots”(lentigines), thinned or fragile skin). In addition, in anotherembodiment, the PDT of photodamaged skin according to the invention isapplied on areas of chronic exposure including the face, ears, neck,backs of the hands, chest, arms and/or legs.

According to another aspect, the invention relates to a kit comprising adevice for implementing a pretreatment as provided above, and acomposition comprising a photosensitizer as described above. This kit isuseful for the implementation of the methods and uses of the presentinvention. In a particular embodiment, the kit comprises an adaptedsandpaper and a composition comprising ALA or esters of ALA such as themethyl ALA ester, hexyl ALA ester and benzyl ALA ester, especiallymethyl ALA ester. In addition, the kit according to the invention maycomprise a sunscreen. The kit of the invention may further comprisesinstructions to follow for implementing the methods and uses of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inflammation vs. response rate (3 months)of AK on the face.

FIG. 2 is a graph showing the mean increased redness the day after PDT.

FIG. 3 is a graph reporting the visual redness 1 day after PDT withdifferent treatment protocols.

FIG. 4 is a graph showing the pain scale after different treatments.

FIG. 5 is a graph showing the cure rate after different treatments.

FIG. 6 is a graph showing the increase in erythema percentage one dayafter treatment with different protocols.

FIG. 7 is a graph showing erythema scale after treatment with differentprotocols.

FIG. 8 is a graph showing the mean photobleaching in the standardtreatment and a different “pulse” treatment.

FIG. 9 is a graph showing the inflammation (erythema)/PpIX formationrelationship.

FIG. 10 is a graph showing the values of fluorescence by IMP andpre-treatment at 405 nm.

FIG. 11 is a graph showing the values of fluorescence by IMP andpre-treatment at 632 nm.

FIG. 12 is a graph showing TEWL detailed by occlusion and pre-treatment.

EXAMPLES Example 1—Assessment of the Efficacy of a Daylight PDT inSubjects with Facial Photodamage

A double-blind randomized-controlled trial has been conducted todetermine whether daylight PDT is an efficient method for treatingphotodamaged skin.

60 Subjects were randomized to receive 1 gram of topical methylaminolevulinate (MAL) applied to the whole face <30 minutes before sunexposure for 2 hours (3 sessions, 2 to 4 weeks apart) or matchingplacebo applied to the whole face <30 minutes before sun exposure for 2hours (3 sessions, 2 to 4 weeks apart) in a double-blind fashion.Neither the investigator nor the patient knew which agent wasadministered, as it was applied by a trained professional nurse. Toenhance product/placebo penetration a subtle abrasion with sandpaper ofthe whole face was performed. Also, a sunscreen (Cetaphil DermacontrolSPF30®) was applied to the entire sun-exposed area including thetreatment area in both groups during daylight-PDT, to avoid sunburn.

Efficacy was evaluated after 1 month of the third (last) daylightsession.

PDT treatment with MAL followed by daylight exposure was found to have asignificantly greater treatment effect than placebo followed by daylightexposure; with the majority of patients of the MAL group having facialimprovement (14 out of 30 subjects) and 11 out of 30 having facialsuccess (p=0.00, Chi² test).

Significant differences were also found in specific photodamagevariables:

Specific photodamage severity scores Metvix Placebo p value Fine linesfailure 6 (20%) 27 (90%) 0.000 improvement 13 (43.3%) 2 (6.7%) sucess 11(36.7%) 1 (3.3%) Mottled pigmentation failure 7 (23.3%) 23 (76.7%) 0.000improvement 18 (60%) 4 (13.3%) sucess 5 (16.7%) 3 (10%) Sallownessfailure 5 (16.7%) 25 (83.3%) 0.000 improvement 9 (30%) 2 (6.7%) sucess16 (53.3%) 3 (10%) Tactile roughness failure 5 (16.7%) 25 (83.3%) 0.000improvement 6 (20%) 2 (6.7%) sucess 19 (63.3%) 3 (10%) Coarse linesfailure 9 (30%) 27 (90%) 0.000 improvement 15 (50%) 3 (10%) sucess 6(20%) 0 (0%) Erythema failure 6 (20%) 25 (83.3%) 0.000 improvement 16(53.3%) 2 (6.7%) sucess 8 (26.7%) 3 (10%)

Pain VAS scores after session 1, 2 and 3 were not significantlydifferent between the two groups, although a little bit more pain wasexperimented by patients in the placebo group after the second session(below table).

Similarly, there were no statistical differences in the effectsexperimented by patients by patients one week after sessions, and insome of these variables no effect was found depending on each session(below table).

Other secondary Metvix Placebo Outcomes (Mean(Median)) (Mean(Median)) PPain VAS score after 0.80 (0) 0.40 (0) >0.05 session 1 Pain VAS scoreafter 0.60 (0) 1.27 (0) >0.05 session 2 Pain VAS score after 1.13 (0)8.77 (0) >0.05 session 3 n (%) n (%) Reaction 1 week after session 1Oozing 0 30 (100%) 30 (100%) * 1 0 (0%) 0 (0%) 2 0 (0%) 0 (0%) 3 0 (0%)0 (0%) Erythema 0 20 (66.7%) 25 (83.3%) >0.05 1 8 (26.7%) 5 (16.7%) 2 2(6.7%) 0 (0%) 3 0 (0%) 0 (0%) Oedema 0 30 (100%) 30 (100%) * 1 0 (0%) 0(0%) 2 0 (0%) 0 (0%) 3 0 (0%) 0 (0%) Desquamation 0 16 (53.3%) 23(76.7%) >0.05 1 9 (30%) 5 (16.7%) 2 5 (16.7%) 2 (6.7%) 3 0 (0%) 0 (0%)Pigmentation 0 29 (96.7) 29 (96.7%) >0.05 1 1 (3.3%) 1 (3.3%) 2 0 (0%) 0(0%) 3 0 (0%) 0 (0%) Vesiculation 0 30 (100%) 30 (100%) * 1 0 (0%) 0(0%) 2 0 (0%) 0 (0%) 3 0 (0%) 0 (0%) Reaction 1 week after session 2Oozing 0 30 (100%) 30 (100%) * 1 0 (0%) 0 (0%) 2 0 (0%) 0 (0%) 3 0 (0%)0 (0%) Erythema 0 19 (63.3%) 22 (73.3%) >0.05 1 11 (36.7%) 8 (26.7%) 2 0(0%) 0 (0%) 3 0 (0%) 0 (0%) Oedema 0 30 (100%) 30 (100%) * 1 0 (0%) 0(0%) 2 0 (0%) 0 (0%) 3 0 (0%) 0 (0%) Desquamation 0 15 (50%) 23(76.7%) >0.05 1 12 (40%) 7 (23.3%) 2 3 (10%) 0 (0%) 3 0 (0%) 0 (0%)Pigmentation 0 29 (96.7%) 30 (100%) >0.05 1 1 (3.3%) 0 (0%) 2 0 (0%) 0(0%) 3 0 (0%) 0 (0%) Vesiculate 0 38 (100%) 30 (100%) * 1 0 (0%) 0 (0%)2 0 (0%) 0 (0%) 3 0 (0%) 0 (0%) Reaction 1 week after session 3 Oozing 030 (100%) 30 (100%) * 1 0 (0%) 0 (0%) 2 0 (0%) 0 (0%) 3 0 (0%) 0 (0%)Erythema 0 23 (76.7%) 26 (86.7%) >0.05 1 7 (23.3%) 4 (13.3%) 2 0 (0%) 0(0%) 3 0 (0%) 0 (0%) Oedema 0 30 (100%) 30 (100%) * 1 0 (0%) 0 (0%) 2 0(0%) 0 (0%) 3 0 (0%) 0 (0%) Desquamation 0 15 (50%) 22 (73.3%) >0.05 112 (40%) 8 (26.7%) 2 3 (10%) 0 (0%) 3 0 (0%) 0 (0%) Pigmentation 0 26(86.7%) 28 (93.3%) >0.05 1 4 (13.3%) 2 (6.7%) 2 3 (10%) 0 (0%) 3 0 (0%)0 (0%) Vesiculation 0 30 (100%) 30 (100%) * 1 0 (0%) 0 (0%) 2 0 (0%) 0(0%) 3 0 (0%) 0 (0%)

Example 2—Comparison of Mechanical Penetration Enhancers onPhotosensitizer Skin Penetration

The effect on the product skin penetration of different mechanicalpenetration enhancement techniques (occlusion, microneedles, ablativefractional laser) has been evaluated.

10 healthy volunteers have been treated according to the followingprotocol:

-   -   pretreatment with either micro-needles (Dermaroller) or ablative        fractional laser (CO₂ laser fraxel repair (SOLTA)), or no        pretreatment;    -   application of Metvix;    -   3 hours of incubation with or without occlusion.    -   Penetration was quantified during incubation using measurement        of photo fluorescence of PpIX at 30 minutes, 1 hour, 2 hours,        and 3 hours after product application.

Both Dermaroller and laser similarly increased Metvix penetration insurface and deeper skin as measured by blue (405 nm) (see FIG. 10 ) andred (632 nm) (see FIG. 11 ) photo fluorescence as compared to nopretreatment without occlusion and no pretreatment with occlusion.

No difference was observed with or with occlusion before 3 hours.

In addition, laser pretreatment was found to be more painful and moreirritant than Dermaroller, and laser pretreatment has more impact inlowering skin barrier function as observed by measuring transepidermalwater loss (see FIG. 12 ).

Therefore, the inventors have surprisingly shown that mechanicalpretreatment with a device such as micro-needle device is as efficientas a laser pretreatment to increase product skin penetration but withless adverse events and is therefore more adapted to the PDT treatmentof photodamaged skin.

Example 3—PDT Procedure Change to Minimize Inflammation in PDT

According to the just mentioned theory it would be preferable to keepPPIX and cellular enzymes away from the extracellular compartment,thereby avoiding inflammation. The purpose of this project is thereforeto keep the PPIX formation within the mitochondria and avoid excessamounts of PPIX to be formed. Simultaneously PPIX should be allowed tobe formed for such a long time that most unnormal cells will beaffected.

So the purpose of PDT is to kill unnormal cells, preferably byapoptosis. The ideal situation would be to keep PPIX inside the cell andto destroy the mitochondria only, thereby inhibiting the ATP formationnecessary for cell functions. That should result in cell death byapoptosis.

One possible way to achieve this would be to give a short 5-MAL pulsetreatment to get a high concentration of 5-MAL in the cells initiallyand then diminish further access to 5-MAL by removing 5-MAL from theskin surface.

This could be done by only exposing the skin to 5-MAL for a short time,after which all 5-MAL is removed from the skin surface. If the right“pulse time” can be found it might ensure high cellular PPIX and lowextracellular PPIX. Excess amounts of PPIX formation during and afterthe end of the treatment would thus be avoided.

The result shows less inflammation with unchanged efficacy and thusmitochondria destruction seems to be the most important factor in PDT.

To estimate the preferable Metvix “pulse time” a separate investigationwas performed (Method B) on 24 healthy volunteers. The pulse time was 20min., 40 min., 60 min., and the conventional 180 min, after which excessamounts of Metvix was removed from the skin. The formation of PPIX after3 hours is seen in FIG. 8 , and the relation to inflammation is seen inFIG. 9 . It is seen that PPIX concentration speeds up between 20-40 min.of “pulse exposure”, and so we have chosen 30 min. as the minimum “pulseexposure” time in the following (Method A) investigation of efficacy andinflammation by this method change. The results are illustrated inColumn 3 in FIGS. 4, 5, 6, and 7 . The procedure change clearlydiminishes inflammation (erythema), without affecting the cure rate(FIG. 5 ). Pain level is not changed. PPIX concentration is clearlylower than for the conventional 3-hour exposure to Metvix (Table 1 andFIG. 8 ).

Methods

Healthy Volunteers

Twenty-four healthy male volunteers of Scandinavian ancestry wereincluded in the study (mean age 30 years, range 20-51). A treatment areawas selected on the inside of both forearms of the volunteer. Eachtreatment area was divided into four minor treatment fields of the size2×5 cm with at least 3 cm between each field using a prefabricatedflexible template. In order to imitate skin lesions all fields were tapestripped 10 times with occlusive dressing before treatment (Tegaderm™Roll, 3M, Glostrup, Denmark).

On the left forearm vehicle Unguentum M was applied to the treatmentfield.

On the right forearm excess amounts of 5-MAL 16% (Metvix®, Photocure,Oslo, Norway) were applied to all four fields of treatment. All fieldswere covered with light-impermeable, occlusive dressing. After 20minutes the dressing was removed from the first field and the excesscream gently wiped off. The field was covered again with a thin piece ofgauze and light impermeable dressing. After additional 20 and 40 minsame procedure was followed with the second and third field. 180 minafter application of 5-MAL and vehicle was removed from all five fields,and the excess cream was gently wiped of the last field. All fields wereilluminated with red light. Illumination was performed with red LEDlight 630 nm peak (Aktilite™ 128; Photocure ASA, Oslo Norway) using atotal light dose of 37 J/cm² given over 9 min. During and afterillumination pain was recorded. The volunteers were equipped with aspecial diary for recording pain in the days after treatment. Fourfollow-up visits were performed at day 1, 2, 3 and 8 after treatment.

PpIX Fluorescence

5-MAL-induced PpIX fluorescence was depicted non-invasively using afluorescence camera (Medeikonos AB, Gothenburg, Sweden). The amount ofPpIX fluorescence was calculated from the photographs by the programMatLab® (MatLab®, MathWorks, Natic, US). The amount of fluorescence wasmeasured before tape stripping and cream application (baseline) andbefore and after illumination.

The photo bleaching is then the difference in PpIX fluorescence (AU)calculated from the pre and post illumination images.

Erythema and Pigmentation

As an indicator of inflammation erythema was measured. The erythema wasassessed by an expert evaluator and measured objectively.

The objective measurements of erythema and pigmentation were performedusing a skin reflectance meter (Optimize Scientific 558, Chromo-Light,Espergaerde, Denmark).

Erythema % and pigmentation % were measured before treatment,immediately before illumination, immediately after illumination, and atthe four follow-up visits.

Pain Score

The volunteers scored their pain every minute during illumination, andrecorded their pain in the diary every hour after illumination on thetreatment day, twice per day the next three days and once a day on thefollowing five days. Since PDT was performed at different times of theday the number of evaluations differed from 3 to 11 the first day. Painwas assessed using a numerical scale ranging from 0 to 10, where 0 is nopain and 10 is worst imaginable pain. To make it easier for the patientsto identify the different treated fields, the dairy was supplied withnumbered drawings of the fields.

Randomizing

The study was designed as an open randomised trial. A statisticaladviser made the randomisation. Since the sequence of treatment durationwas predefined, randomization was only determining which of the fourtreatment fields should be the first.

Statistics

The sample size was calculated on the bases of data from the literature.We set the minimal clinical relevant difference to 8.8% (50% of theearlier found 17.6%) and choose a power of 0.80 and a significance levelof 0.05, 22 volunteers should be included.

To identify differences in pain score, erythema % and pigmentation %between the treatment fields we used Wilcoxon Signed Ranked Test, sinceall results were paired.

For all calculations a p-value<0.05 was considered statisticalsignificant. All analyses were performed with PASW Statistics 19.0 forWindows (SPSS Inc, Chicago, Ill., USA).

The invention claimed is:
 1. A method of treating actinic keratosis in ahuman subject, the method comprising: (a) subjecting actinic keratosislesions on skin of the subject to a curettage mechanical pretreatment,(b) administering to the pretreated lesions a composition comprising5-aminolevulinic acid methyl ester or a pharmaceutically acceptable saltthereof, (c) exposing the lesions to natural light for 0.5 to 3 hours,and (d) optionally removing the composition.
 2. The method according toclaim 1, wherein the composition is removed after exposing the skin tothe natural light.
 3. The method according to claim 1, wherein thecomposition is not removed after exposing the skin to the natural light.4. The method according to claim 1, wherein the composition is in theform of a cream.
 5. The method according to claim 1, wherein the naturallight is sunlight.
 6. The method according to claim 1, wherein the humansubject is female.
 7. The method according to claim 1, wherein the humansubject is male.
 8. The method according to claim 1, wherein themechanical pretreatment removes or perforates the skin stratum corneum.9. The method according to claim 1, wherein the lesions are located onthe skin of the face and/or scalp.
 10. The method according to claim 1,wherein the lesions are premalignant.
 11. The method according to claim1, wherein the lesions are malignant.
 12. The method according to claim1, further comprising administering a sunscreen to the skin that will beexposed to the natural light prior to the mechanical pretreatment. 13.The method according to claim 1, wherein the composition is administeredto the pretreated lesions 4 to 35 minutes before exposing the lesions tonatural light.
 14. The method according to claim 1, wherein thecomposition is administered to the pretreated lesions about 30 minutesbefore exposing the lesions to natural light.
 15. The method accordingto claim 1, wherein the lesions are exposed to natural light for 2 to 3hours.
 16. The method according to claim 1, wherein (a)-(d) are repeatedone or more times 2-4 weeks apart.
 17. The method according to claim 1,wherein (a)-(d) are repeated one or more times at least two weeks apart.18. The method according to claim 1, wherein (a)-(d) are repeated twoweeks later.
 19. The method according to claim 1, wherein (a)-(d) arerepeated twice more, two weeks apart.
 20. The method according to claim1, wherein the composition comprises a pharmaceutically acceptable saltof 5-aminolevulinic acid methyl ester.